Pre-hatching fluoxetine-induced neurochemical, neurodevelopmental, and immunological changes in newly hatched cuttlefish.

Embryonic and early postembryonic development of the cuttlefish Sepia officinalis (a cephalopod mollusk) occurs in coastal waters, an environment subject to considerable pressure from xenobiotic pollutants such as pharmaceutical residues. Given the role of serotonin in brain development and its interaction with neurodevelopmental functions, this study focused on fluoxetine (FLX), a selective serotonin reuptake inhibitor (SSRI, antidepressant). The goal was to determine the effects of subchronic waterborne FLX exposure (1 and 10 µg L(-1)) during the last 15 days of embryonic development on neurochemical, neurodevelopmental, behavioral, and immunological endpoints at hatching. Our results showed for the first time that organic contaminants, such as FLX, could pass through the eggshell during embryonic development, leading to a substantial accumulation of this molecule in hatchlings. We also found that FLX embryonic exposure (1 and 10 µg L(-1)) (1) modulated dopaminergic but not serotonergic neurotransmission, (2) decreased cell proliferation in key brain structures for cognitive and visual processing, (3) did not induce a conspicuous change in camouflage quality, and (4) decreased lysozyme activity. In the long term, these alterations observed during a critical period of development may impair complex behaviors of the juvenile cuttlefish and thus lead to a decrease in their survival. Finally, we suggest a different mode of action by FLX between vertebrate and non-vertebrate species and raise questions regarding the vulnerability of early life stages of cuttlefish to the pharmaceutical contamination found in coastal waters.

Physiological perturbations in juvenile cuttlefish Sepia officinalis induced by subchronic exposure to dissolved zinc.

Although cephalopod early life stage development often occurs in coastal areas where contamination is real and continuous, the physiological perturbations induced by contaminants have been rarely investigated. This study focused on the Zn as it is one of the trace metals the most concentrated in coastal waters, worldwide. As Zn-tolerance limits were unknown in juvenile Sepia officinalis, the aim of this study was to estimate the threshold inducing mortality during the 2-first weeks post-hatching, and to determine its sensitivity using digestive and immune enzymatic assays, as well as growth and behavior follow-up during the first 5weeks post-hatching. Our study highlighted a Zn-mortality threshold lying between 185 and 230µgl(-1), and growth reductions occurring after 5-week at 108µgl(-1) and above, associated with enzymatic perturbations. These results underline a relatively important sensitivity of juvenile cuttlefish to Zn, pointed out by a wide diversity of biomarkers.

Trace metal concentrations in post-hatching cuttlefish Sepia officinalis and consequences of dissolved zinc exposure.

In this study, we investigated the changes of 13 trace metal and metalloid concentrations (i.e. Ag, As, Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb, Se, V, Zn) and their subcellular fractionation in juvenile cuttlefish Sepia officinalis reared in controlled conditions between hatching and 2 months post-hatching. In parallel, metallothionein concentrations were determined. Our results highlighted contrasting changes of studied metals. Indeed, As and Fe concentrations measured in hatchlings suggested a maternal transfer of these elements in cuttlefish. The non-essential elements Ag and Cd presented the highest accumulation during our study, correlated with the digestive gland maturation. During the 6 first weeks of study, soluble fractions of most of essential trace metals (i.e. Co, Cr, Cu, Fe, Se, Zn) slowly increased consistently with the progressive needs of cuttlefish metabolism during this period. In order to determine for the first time in a cephalopod how metal concentrations and their subcellular distributions are impacted when the animals are trace metal-exposed, we studied previously described parameters in juveniles exposed to dissolved Zn at environmental (i.e. 50 µg l(-1)) and sublethal (i.e. 200 µg l(-1)) levels. Moreover, oxidative stress (i.e. glutathione S-transferase (GST), superoxide dismutase (SOD) and catalase activities, and lipid peroxidation (LPO)) was assessed in digestive gland and gills after 1 and 2 months exposures. Our results highlighted no or low ability of this stage of life to regulate dissolved Zn accumulation during the studied period, consistently with high sensitivity of this organism. Notably, Zn exposures caused a concentration-dependent Mn depletion in juvenile cuttlefish, and an increase of soluble fraction of Ag, Cd, Cu without accumulation modifications, suggesting substitution of these elements (i.e. Mn, Ag, Cd, Cu) by Zn. In parallel, metallothionein concentrations decreased in individuals most exposed to Zn. Finally, no perturbations in oxidative stress management were detected in gills, whereas modifications of GST, SOD and catalase activity levels were recorded in digestive gland, resulting in an increase of LPO content after a 6-week exposure to low Zn concentration. Altogether, these perturbations are consistent with previously described high sensitivity of juvenile cuttlefish towards Zn. Our results underlined the need to study deeply contamination impact on this animal at this stage of life.

Hemocyte morphology and phagocytic activity in the common cuttlefish (Sepia officinalis).

Little is known about the immune system of cephalopods, in spite of their many highly derived characters within the molluscan clade, including a vertebrate-like high-pressure closed circulatory system. Further the economic importance of cephalopod fisheries, potential for aquaculture, and use as ecotoxicology models demand a thorough understanding of their immune system. In this study, we present a comprehensive characterization of hemocytes in the common cuttlefish Sepia officinalis. Cytological stainings, electron microscopy- and flow cytometry-observations highlight a single granulocyte population with various densities of eosinophilic granules and unstained vesicles. These hemocytes contain acid phosphatase-, lysozyme- and proPO system enzymes, and have high activity in bead phagocytosis assays. Interestingly, bead pre-incubation in plasma results in time-dependent aggregation perhaps resulting from hemocyanin-coating, and decrease in phagocytosis. This study provides the basis for understanding hemocyte-mediated immunity in the common cuttlefish, and essential background for future studies on cephalopod immunity.

Cephalopod culture: current status of main biological models and research priorities.

A recent revival in using cephalopods as experimental animals has rekindled interest in their biology and life cycles, information with direct applications also in the rapidly growing ornamental aquarium species trade and in commercial aquaculture production for human consumption. Cephalopods have high rates of growth and food conversion, which for aquaculture translates into short culture cycles, high ratios of production to biomass and high cost-effectiveness. However, at present, only small-scale culture is possible and only for a few species: the cuttlefish Sepia officinalis, the loliginid squid Sepioteuthis lessoniana and the octopuses Octopus maya and O. vulgaris. These four species are the focus of this chapter, the aims of which are as follows: (1) to provide an overview of the culture requirements of cephalopods, (2) to highlight the physical and nutritional requirements at each phase of the life cycle regarded as essential for successful full-scale culture and (3) to identify current limitations and the topics on which further research is required. Knowledge of cephalopod culture methods is advanced, but commercialization is still constrained by the highly selective feeding habits of cephalopods and their requirement for large quantities of high-quality (preferably live) feed, particularly in the early stages of development. Future research should focus on problems related to the consistent production of viable numbers of juveniles, the resolution of which requires a better understanding of nutrition at all phases of the life cycle and better broodstock management, particularly regarding developments in genetic selection, control of reproduction and quality of eggs and offspring.

Prophenoloxidase system, lysozyme and protease inhibitor distribution in the common cuttlefish Sepia officinalis.

The immune system of cephalopods remains poorly understood. The aim of this study was to determine the specific activity of immune enzymes in epithelial barriers, circulatory and digestive systems of the common cuttlefish Sepia officinalis. Three enzyme groups with putative functions in immunity were investigated: phenoloxidases (POs), lysozymes and protease inhibitors (PIs). Consistent with a role in immunity, highest PO activities were found in the integument as well as the respiratory and circulatory organs under zymogenic (proPO) and active form. Surprisingly, high PO activities were also found in the digestive gland and its appendages. Similarly, high lysozyme activities were detected in the integument and circulatory organs, but also in the posterior salivary glands, highlighting the implication of this antibacterial enzyme group in most tissues exposed to the environment but also within the circulatory system. Albeit highest in digestive organs, the ubiquitous detection of PI activity in assayed compartments suggests immune function(s) in a wide range of tissues. Our study reports proPO/PO, lysozyme and PI distributions in S. officinalis body compartments for the first time, and thus provides the fundamental basis for a better understanding of the humoral immune system in cephalopods as well as invertebrates.

Responses of primary cultured haemocytes from the marine gastropod Haliotis tuberculata under 10-day exposure to cadmium chloride.

Among metals, cadmium, a non-essential element, is an important pollutant that is released into aquatic environments. Due to its persistence and bioaccumulation, this metal has been shown to exert immunological effects on organisms. The objective of the present study was to investigate the in vitro effects of cadmium chloride using a haemocyte primary culture from the European abalone, Haliotis tuberculata. Most studies have maintained viable haemocytes in vitro for periods ranging from several hours to several days during acute exposures. Few investigations have reported the effects of metals using longer in vitro exposures, which are more realistic with regard to mimicking environmental conditions. In this study, we exposed abalone haemocytes to concentrations from 0.5 to 50,000 µgL(-1) of CdCl2 for 10 days. The effects of cadmium chloride were reflected in a significant decrease in the number of viable cells and morphological modifications in a concentration-dependent manner beginning at a concentration of 500 µgL(-1) as well as in some physiological processes, such as phagocytotic activity and the number of lysosome-positive cells. In contrast, phenoloxidase (PO) activity and reactive oxygen species (ROS) production were increased beginning at a concentration of 5 µgL(-1), which is consistent with environmental concentrations in polluted sites. For PO activity and ROS production, maximally 9-fold and 130% inductions, respectively, were recorded under the highest dose. These results thus indicate that cadmium chloride alters immune parameters of abalone haemocytes and that the long-term (10 days) primary culture system used here represents a suitable, sensitive in vitro model for assessing cytotoxic responses.

[The modalities of the sexual cycle of Octopus vulgaris in the southern Moroccan Atlantic (Tantan, Boujdour)]. / Les modalités du cycle sexuel du poulpe Octopus vulgaris du Sud marocain (Tantan, Boujdour).

The reproduction of Octopus vulgaris between Tan Tan and Boujdour is studied. The samples used are sorted monthly from commercial catches of coastal trawlers operating in this area from December 2001 to May 2003. The study shows that sexual maturity is in advance for males than for females. Two spawning periods are made out by the follow-up of the RGS: a spring period from March to July, and an autumnal period, which is less intense, between September and October. The division of spermatogonia and spermatocytes reaches its maximal intensity in July and December, whereas the spermiogenesis is active until the time of mating. The vitellogenesis starts in mid-December and extends until the following spawning period. This reveals a later gonadic development for females than for males; approximately seven months for females and three months for males.

Variation of heavy metal concentrations (Ag, Cd, Co, Cu, Fe, Pb, V, and Zn) during the life cycle of the common cuttlefish Sepia officinalis.

The developmental changes in the concentration of 8 essential and non-essential heavy metals (Ag, Cd, Cu, Co, Fe, Pb, V, Zn) in the tissues (digestive gland, cuttlebone and whole animal) of the common cuttlefish Sepia officinalis collected in the bay of the river Seine were monitored from the end of the embryogenesis until the adult reproductive stage. Compared to embryos, juveniles after hatching displayed much higher concentrations of Ag, Cu, Fe and Zn, suggesting an efficient incorporation from seawater. Conversely, the amounts of Cd, Pb and V in hatchlings remained constant suggesting that these metals are barely bioavailable for juveniles. Once the juveniles start to feed, the digestive gland appears to play a major role in the storage of all metals. After only one month of benthic life, the digestive gland already contains up to 90% of the total metal body burden, indicating that it plays a major role in the storage and presumed detoxification of the selected metals. Metal concentrations in the digestive gland increase in a logarithmic fashion with age during the entire life of cuttlefish, except for Ag, which decreases as soon as cuttlefish migrate to open sea. This strongly suggests that (1) Ag is excreted from the digestive gland in relation to presumably lower exposure in less contaminated environments compared to coastal waters and (2) the digestive gland of cephalopods could be a very good indicator of Ag contamination in the marine environment.

Androgenic effect of Mondia whitei roots in male rats.

AIM: To determine the effect of the aqueous extract of Mondia whitei (Periplocaceae) roots on testosterone production and fertility of male rats. METHODS: Adult male Wistar rats were used. In the acute study, 20 rats were randomly divided into 5 groups of 4 animals each. Four treated groups were administered orally a single dose of Mondia whitei (400 mg/kg) and the controls received a similar amount of distilled water. One group of animals were sacrificed by cervical dislocation 1, 2, 4 and 6 h after treatment, respectively. The controls were sacrificed at 6 h. Testicular testosterone was determined by radioimmunoassay. In the chronic study, 28 rats were divided at random into 4 groups of 7 animals each: Groups 1, 2 and 3 were given orally the plant extract (400 mg.kg(-1).day(-1)) for 2, 4 and 8 days, respectively. The animals of Groups 1 and 2 were sacrificed 24 hours after the last dosing. The controls (Group 4) received the same amount of distilled water for 8 days. The fertility was assessed only in Groups 3 and 4 and after that, the animals were sacrificed and the epididymal sperm density, the serum testosterone and the testicular testosterone and 17 beta-estradiol were assayed. The serum, testicular and epidydimal protein contents were also determined. RESULTS: In the acute treatment groups, the serum and testicular concentrations of testosterone remained unchanged at all the time points. Chronic treatment for 8 days induced a significant increase in the testicular weight, the serum and testicular testosterone, the testicular protein content and the sperm density (P < 0.05-0.01), but did not affect the accessory gland weights, the serum protein contents, the testicular concentration of 17beta -estradiol and the fertility compared to the controls. CONCLUSION: Mondia whitei root extract possesses an androgenic property.